CN111500705B - IgAN intestinal flora marker, igAN metabolite marker and application thereof - Google Patents
IgAN intestinal flora marker, igAN metabolite marker and application thereof Download PDFInfo
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Abstract
The invention provides an IgAN intestinal flora marker, an IgAN metabolite marker and application thereof. The gAN marker of intestinal flora comprises at least one of Streptococcus (Streptococcus), enterococcus (Enterococcus) and Bacteroides (Bacteroides). The inventor collects the fecal samples of the IgAN patients and normal people, screens out the intestinal microorganisms with higher correlation with the IgAN from the microorganisms in the fecal samples, and can effectively determine whether the subjects have the disease risk of the IgAN or make certain judgment on the effect of the treatment scheme of the IgAN patients by using the intestinal microorganism or the intestinal microorganisms as biomarkers.
Description
Technical Field
The invention relates to the technical field of nephropathy, in particular to an IgAN intestinal flora marker, an IgAN metabolite marker and application thereof.
Background
IgA nephropathy (IgAN) is the most common disease with chronic renal function impairment in primary glomerulonephritis. IgAN is the leading cause of chronic renal failure in China. However, the pathogenesis of IgAN is not clear, and is related to many factors such as pathogen infection, mucosal immunity, cellular immunity, and inflammatory mediators. At the same time, there is increasing research evidence that genetic factors play a non-negligible role in the development of IgAN.
Because the pathogenesis of IgAN is difficult to discern, a convenient, rapid, and effective method for diagnosing IgAN is still lacking. IgAN is usually diagnosed clinically by a combination of several methods, such as measurement of IgA levels in the serum of a patient, examination of urine to determine the level of proteinuria, measurement of Glomerular Filtration Rate (GFR) and Renal Biopsy (Renal Biopsy). Among the diagnostic gold criteria for IgAN are kidney biopsies. However, kidney biopsy procedures are complex, the procedure is traumatic and painful, and other complications are most likely to arise to some extent. Therefore, a convenient and fast method for guiding the diagnosis and treatment of clinical IgAN is needed.
The intestinal flora is the general term of various microorganisms in human intestinal tracts, and the total number of the microorganisms in the flora is 100 trillion and is about 10-20 times of the total number of human cells, so the intestinal flora is called as a 'second genome' of human and is very important for the health of the human body. On one hand, they can affect the physiological activities of the host by influencing the metabolism and gene expression of the host, and directly participate in various aspects of human digestion, nutrient absorption, energy supply, fat metabolism, immunoregulation, disease resistance and the like. On the other hand, an imbalance in intestinal flora is considered to be closely related to many inflammatory diseases in humans, and can cause low-level systemic chronic inflammation in the host. More and more studies have shown that various diseases in humans are closely related to the function of the intestinal flora, such as Inflammatory Bowel Disease (IBD), liver disease, type ii diabetes, tumors, etc. Current clinical practice shows that IgAN is the most commonly diagnosed disease in patients with inflammatory bowel disease. This may suggest that the gut flora also plays an important role in the development of IgAN. Therefore, there is a need to develop specific IgAN biomarkers for intestinal flora to provide a new scientific approach for rapid and sensitive diagnosis of diseases.
Disclosure of Invention
The present invention is directed to solving at least one of the problems of the prior art. Therefore, the invention provides an IgAN intestinal flora marker, an IgAN metabolite marker and application thereof.
In a first aspect, an embodiment of the present invention provides an IgAN intestinal flora marker including at least one of Streptococcus (Streptococcus), enterococcus (Enterococcus), bacteroides (Bacteroides).
The intestinal flora marker provided by the embodiment of the invention at least has the following beneficial effects:
the inventor collects fecal samples of IgAN patients and normal people, screens out intestinal microorganisms with high correlation with IgAN from the microorganisms in the fecal samples, and can effectively determine whether a subject has the risk of the IgAN or not by using the intestinal microorganism or the intestinal microorganisms as biomarkers or make certain judgment on the effect of a treatment scheme of the IgAN patients.
In a second aspect, an embodiment of the present invention provides an IgAN metabolite marker, including at least one of bilirubin (bilirubin), trimethoprim (trimethoprim), stearamide (stearamide), phenylalanine (phenylalanine), cis-9,10-epoxystearic acid (cis-9,10-epoxystearic acid), lysophosphatidylethanolamine 17 (PE lyso 17.
The metabolite marker of the embodiment of the invention has at least the following beneficial effects:
the inventors have found that specific metabolites are significantly different between patients and normal population by collecting blood samples of IgAN patients and normal population, and the metabolites with significant content difference can be used as biological markers of IgAN for diagnosis or prognosis evaluation.
In a third aspect, an embodiment of the present invention provides a use of the above IgAN intestinal flora marker or IgAN metabolite marker in the preparation of an IgAN diagnostic and/or prognostic reagent. Obtaining related information of the intestinal flora by detecting a fecal sample of a subject, and comparing the content of the marker of the intestinal flora with a preset threshold value to further judge whether the subject is in a disease risk with IgAN or make a reasonable and effective judgment on the curative effect of a treatment scheme of the patient; or the content of the metabolite marker in the serum, urine, feces and other samples of the subject is compared with a preset threshold value by detecting the related information of the metabolite in the serum, urine, feces and other samples of the subject, so as to judge whether the subject is in the risk of the disease with the IgAN or reasonably and effectively judge the curative effect of the treatment scheme of the patient.
In a fourth aspect, the present invention provides a use of the above IgAN intestinal flora marker or IgAN metabolite marker in screening for a drug for preventing and/or treating IgAN. Judging whether the medicament passes the screening or not by detecting whether the content of the intestinal flora marker in the excrement sample is reduced below a threshold value or not after the periodic medicament use of the subject is finished; or judging whether the drug passes the screening by detecting whether the content of the metabolite markers in the serum, urine, excrement and other samples of the subjects is reduced below a threshold value after the periodic drug use is finished.
The content and the threshold value may be the content or abundance of a single or multiple markers, or may be relative values obtained by modeling, analyzing and processing the content or abundance of multiple markers.
In a fifth aspect, an embodiment of the present invention provides a kit comprising reagents for detecting the amount of markers, which are the above-mentioned markers for the gut flora of IgAN and/or the markers for metabolites of IgAN. The effective use of Streptococcus (Streptococcus), enterococcus (Enterococcus), bacteroides (Bacteroides) in fecal samples of IgAN patients and normal healthy control groups, bilirubin (bilirubin), trimethoprim (trimethoprim), stearamide (stearamide), phenylalanine (phenylalanine), cis-9,10-epoxystearic acid (cis-9,10-epoxystearic acid), lysophosphatidylethanolamine 17 (PE lyso 17 0) in fecal samples and serum samples can be determined by detecting one or more of the above markers.
In a sixth aspect, an embodiment of the present invention provides a method for assessing risk of IgAN, the method comprising the steps of:
(1) Obtaining the content of the IgAN intestinal flora marker and/or the IgAN metabolite marker in a subject sample;
(2) Comparing the content with a preset threshold value, and judging the IgAN risk according to a comparison result;
this method is not suitable for the diagnosis and/or treatment of diseases.
Wherein the IgAN risk refers to whether the subject has IgAN, and may further include the risk of the disease according to the comparison with the threshold; or to the prognosis of an IgAN patient; or the effect of the drug to be screened on the IgAN model.
According to the method of some embodiments of the present invention, the content of the marker of intestinal flora can be obtained by performing 16S sequencing on the sample; the content of the metabolite marker can be obtained by performing chromatographic analysis, mass spectrometric detection and the like on the sample.
According to the method of some embodiments of the present invention, the subject may be judged to have IgAN, or the prognosis of the IgAN patient is poor, or the drug does not meet the screening condition, if at least one of the following conditions is met:
(1) The content of Streptococcus (Streptococcus) is higher than a threshold value;
(2) The content of Enterococcus (Enterococcus) is lower than the threshold value;
(3) The content of Bacteroides (Bacteroides) is higher than a threshold value;
(4) The content of bilirubin (bilirubin) is higher than a threshold value;
(5) The content of trimethoprim (trimethoprim) is lower than a threshold value;
(6) The content of stearamide (stearamide) is lower than a threshold value;
(7) The content of phenylalanine (phenylalkane) is higher than a threshold value;
(8) The content of lysophosphatidylethanolamine 17 (PE lyso 17).
In a seventh aspect, an embodiment of the present invention provides a computer-readable storage medium storing computer-executable instructions for performing the above-mentioned method. By performing the above method, it is possible to accurately and efficiently determine whether a subject has IgAN, or the prognosis of an IgAN patient.
In an eighth aspect, an embodiment of the present invention provides a system for assessing a subject's risk of IgAN, the system comprising:
a detection device for determining the content of the IgAN intestinal flora marker and/or the IgAN metabolite marker in the sample of the subject;
and the comparison device is used for comparing the content with a preset threshold value and judging the IgAN risk according to the comparison result.
According to some embodiments of the invention, the system, wherein the detection means comprises:
a nucleic acid isolation device for isolating a nucleic acid sample from a sample of a subject;
and the sequencing device is used for detecting the content of the IgAN intestinal flora marker in the nucleic acid sample.
Drawings
FIG. 1 shows the results of clustering analysis of fecal metabolites of IgAN patients and healthy control groups according to one embodiment of the present invention.
FIG. 2 shows the result of cluster analysis of serum metabolites of IgAN patients and healthy control groups according to one embodiment of the present invention.
FIG. 3 is the metabolic pathway analysis results of fecal metabolites of IgAN patients and healthy controls according to one embodiment of the present invention.
FIG. 4 shows the results of metabolic pathway analysis of serum metabolites of IgAN patients and healthy control groups according to one embodiment of the present invention.
FIG. 5 is a ROC curve for bilirubin in accordance with one embodiment of the present invention.
FIG. 6 is a ROC curve for trimethoprim according to one embodiment of the present invention.
Fig. 7 is a ROC curve for stearamide according to one embodiment of the present invention.
FIG. 8 is a ROC curve for phenylalanine according to one embodiment of the present invention.
FIG. 9 is a ROC curve for cis-9,10-epoxystearic acid, an embodiment of the present invention.
FIG. 10 is a ROC curve of lysophosphatidylethanolamine 17.
Fig. 11 is a schematic block diagram of a system for assessing a subject's risk of IgAN according to yet another embodiment of the present invention.
Detailed Description
The concept and technical effects of the present invention will be clearly and completely described below in conjunction with the embodiments to fully understand the objects, features and effects of the present invention. It is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments, and those skilled in the art can obtain other embodiments without inventive effort based on the embodiments of the present invention, and all embodiments are within the protection scope of the present invention.
Example 1
Intestinal flora marker screening
The project is examined by medical ethics committee, all volunteers obtain informed consent and sign consent.
1. Collection of samples
The experiment was divided into two groups, 30 patients with IgAN diagnosed by renal biopsy and 30 normal healthy controls.
All selected IgAN patients are matched with the basic conditions of normal healthy control group, such as sex and age of volunteers, and do not have systemic lupus erythematosus, chronic liver diseases, malignant tumors, diabetes, chronic gastrointestinal diseases, nervous system diseases and other autoimmune diseases. All selected volunteers were not treated with glucocorticoid and immunosuppressant for at least three months, were not treated with any antibiotic for one month, and were not eaten with probiotic foods such as yogurt for one month.
Collecting fecal samples of two groups of people before breakfast, sampling according to requirement of fecal collection system of Sarstedt, germany, mixing feces uniformly with sterile spatula, taking 1g, storing in sterile freezing tube, and storing at-80 deg.C.
2.16S sequencing
2.1 extraction of DNA
Stool samples (1 g) were taken, placed on ice and stool DNA extracted according to the instructions of the E.Z.N.A. stool DNA Kit (Omega Bio-tek, norcross, GA, U.S.).
2.2 library construction and sequencing
The extracted DNA sample was diluted to 1 ng/. Mu.L with sterile water, and PCR amplification was carried out using 16963S rDNA V3-V4 region-specific primers using the diluted genomic DNA as a template.
The amplification primers were as follows:
341F：CCTACGGGNGGCWGCAG(SEQ ID No.1)；
806R：GGACTACHVGGGTATCTAAT(SEQ ID No.2)。
the amplification procedure was: at 95 ℃ for 2min;98 ℃,10s,62 ℃,30s,68 ℃,30s,27 cycles; 68 ℃ for 10min.
Detecting the PCR amplification product by agarose gel electrophoresis, and recovering the target band. For use
Constructing a library by using a DNA PCR-Free Sample Preparation Kit, quantifying the constructed library by using the Qubit and the Q-PCR, and sequencing and analyzing a Sample by using a high-throughput sequencing platform Illumina HiSeq2500 after the library is qualified. Each sample yielded no less than 1 ten thousand tags or 100M data.
3. Data analysis
3.1 OTU annotation and abundance analysis
And filtering the original data (Raw reads) to obtain Effective data (Effective Tags). Species annotation was then performed and clustered into OTUs (Operational Taxonomic Units) and species classification was then done by OTU annotation (species annotation results based on the Reads included).
3.2 diversity analysis
The Alpha diversity of the samples was assessed by various indices. The differences in microbial community structure between different samples were assessed by calculating the distance between Bray curis, weighted Unifrac and Unweighted Unifrac from OTU abundance information for the samples. Based on the distance matrix, the results are analyzed by multivariate statistical method, and the differences of microbial community structures among samples and the contribution differences of different classifications to the samples, namely Beta Diversity, are further mined.
3.3 differential identification of specific OTUs
Obtaining the composition and absolute abundance distribution table of each sample OTU on different classification levels, and comparing and testing the sequence quantity difference of each classification unit of the specified classification level between an IgAN patient group and a normal healthy control group through Metastatts statistical algorithm;
and obtaining the composition and relative distribution table of each sample OTU on different classification levels, and obtaining corresponding intestinal flora markers by LEfSe analysis and Metastatts combination. The results are shown in table 1 below, in which HC and IgAN represent normal healthy control groups and IgAN patient groups, respectively.
TABLE 1 Metastats and LefSe analysis results of intestinal microorganisms of two groups of subjects at different classification levels
As can be seen from the table, at this classification level, the P values of microorganisms of three genera of Streptococcus (Streptococcus), bacteroides (Bacteroides) and Enterococcus (Enterococcus) were all less than 0.05, while the absolute values of LDA were all greater than 2, indicating that the intestinal microorganisms of these three genera were significantly different in the population of IgAN patients from the normal healthy control group. Therefore, the intestinal microorganisms of the three genera can be used for identifying whether the subject has the IgAN disease, or evaluating the prognosis level of the IgAN patient, or realizing a certain screening function on the IgAN medicament.
Example 2
Metabolite marker screening
The project is examined by medical ethics committees, all volunteers obtain informed consent and sign consent.
1. Obtaining a sample
1.1 sample Collection
The experiment was divided into two groups, 30 patients with IgAN diagnosed by renal biopsy and 30 normal healthy controls.
All selected IgAN patients are matched with the basic conditions of normal healthy control group, such as sex and age of volunteers, and do not have systemic lupus erythematosus, chronic liver diseases, malignant tumors, diabetes, chronic gastrointestinal diseases, nervous system diseases and other autoimmune diseases. All selected volunteers were not treated with glucocorticoid and immunosuppressant for at least three months, were not treated with any antibiotic for one month, and were not eaten with probiotic foods such as yogurt for one month.
Venous blood of participants was collected by vacuum EDTA blood collection tubes before breakfast, and after centrifuging the blood samples at 4 ℃ and 2200g for 5min, plasma samples were obtained and stored at-80 ℃ for further use. In addition, fecal samples of two groups of people were collected simultaneously, sampled according to the requirements of the fecal collection system of Sarstedt, germany, using a sterile spatula to mix the fecal evenly, 1g was taken and stored in a sterile cryovial and stored to-80 ℃.
1.2 Pre-treatment of plasma samples
Transferring 100 mu L of sample into an EP tube, adding 300 mu L of methanol, adding 10 mu L of internal standard, carrying out vortex mixing for 30 seconds, and carrying out ice-water bath ultrasound for 10min; standing for 1h at the temperature of minus 20 ℃; centrifuging the sample at 13000rpm for 15min at 4 ℃; carefully take out 200. Mu.L of the supernatant into a polypropylene test tube (a sample bottle with an inner cannula) special for high performance liquid chromatography mass spectrometry, take 20. Mu.L of each sample to mix into a Quality Control (QC) sample, and then take 200. Mu.L of the sample for on-machine detection.
1.3 Mass Spectrometry
And (3) using a ThermoFisher scientific UPLC-QE-MS mass spectrometer, adopting an ESI ion source, adjusting parameters of a chromatographic mass spectrometer, selecting a proper sample introduction amount, and obtaining non-targeted metabonomics data of the plasma.
1.4 data processing
And preprocessing the lower machine mass spectrum data. And converting the mass spectrum original data into an mzML format by using ProteWizard software, and then performing retention time correction, peak identification, peak extraction, peak integration, peak alignment and the like by using XCMS. An intensity information table containing mass-to-charge ratio, retention time and intensity information for each peak was obtained.
The metabolites were identified by matching secondary mass spectral fragments of the metabolites using a metabolite spectral database (HMDB, http:// www.hmdb.ca; METLIN, http:// METLIN. Script. Edu/index. Php; massbank, http:// www.massbank.jp/etc.).
Training and testing sets of age, gender matched stool and serum samples, respectively, consisting of IgAN patients and normal healthy controls were established. The clustering condition of the IgAN patient metabolites is observed by adopting methods of Principal Component Analysis (PCA) and partial least squares-discriminant analysis (PLS-DA), differential metabolites between the IgAN patient and a normal healthy control group are screened out by adopting a PLS-DA model in a training set, clustering analysis is carried out by MetabioAnalyst 3.0, pathway analysis of the differential metabolites is completed, and the differential metabolites identified in the training set are verified by an ROC curve in a testing set.
The results are shown in FIGS. 1 to 10. Wherein, fig. 1 and fig. 2 are the results of clustering analysis of fecal and serum metabolites of IgAN patients/healthy controls, respectively. The results of the metamalyst 3.0 cluster analysis are presented as a heat map from which it can be seen that there are six common significant differential metabolites in the stool and serum samples (bilirubin, trimethoprim, stearamide, phenylalanine, cis-9,10-epoxystearic acid, lysophosphatidylethanolamine 17 0), where bilirubin, phenylalanine levels in IgAN patients are up-regulated differentially in both samples and trimethoprim, stearamide, lysophosphatidylethanolamine 17 are down-regulated differentially in both samples.
Fig. 3 and 4 are metabolic pathway analysis results of fecal and serum metabolites of an IgAN patient/healthy control group according to an embodiment of the present invention. As shown in fig. 3 and 4, the abscissa influence (Impact) indicates the importance of the metabolic pathway, i.e., the differential metabolites hit nodes of the metabolic pathway, the larger the Impact indicates the larger the number of hit nodes, and the ordinate-lnP indicates the significance level of the enrichment analysis of the metabolic pathway, and the larger the-lnP value, the higher the significance. As can be seen from the figure, the fecal samples of IgAN patients are mainly involved in arachidonic acid metabolism, aminoacyl-tRNA biosynthesis, phenylalanine metabolism, lysine biosynthesis, lysine degradation, and the serum samples of IgAN patients are mainly involved in pyruvate metabolism, phenylalanine metabolism, arginine and proline metabolism, tryptophan metabolism.
FIGS. 5-10 show ROC curves for bilirubin, trimethoprim, stearamide, phenylalanine, cis-9,10-epoxystearic acid, lysophosphatidylethanolamine 17. As can be seen from the figure, the AUC values of the 6 metabolites are respectively 0.75, 0.79, 0.66, 0.84 and 0.65, and the results show that the diagnostic values of the 6 metabolites are moderate or highly reliable and have higher application values.
Example 3
Fig. 11 is a schematic composition diagram of a system for assessing a subject's risk of IgAN according to yet another embodiment of the present invention. Referring to fig. 11, the present embodiment provides a system for assessing a subject's risk of IgAN, the system comprising: a nucleic acid isolation apparatus 100 for isolating a nucleic acid sample from a stool sample of a subject; a sequencing device 110 for detecting the markers of the IgAN intestinal flora in the nucleic acid sample: the contents of Streptococcus (Streptococcus), enterococcus (Enterococcus) and Bacteroides (Bacteroides); and the comparison device 120 is configured to compare the content of the markers with a preset threshold, and determine the risk of IgAN according to the relative sizes of the detected content of the three markers and the threshold.
Example 4
The present embodiments provide a computer-readable storage medium having stored thereon computer-executable instructions for performing a method comprising:
(1) Obtaining the content of the IgAN intestinal flora marker and/or the IgAN metabolite marker in a subject sample;
(2) Comparing the content with a preset threshold, judging whether the subject has IgAN or not according to the comparison result, judging the prognosis condition of the IgAN patient, or screening the medicament.
The judging method in the step 2 comprises the following steps:
the subject may be judged to have IgAN, or the prognosis of the IgAN patient is poor, or the drug does not meet the screening criteria, if at least one of the following conditions is met:
(1) The content of Streptococcus (Streptococcus) is higher than a threshold value;
(2) The content of Enterococcus (Enterococcus) is lower than the threshold value;
(3) The content of Bacteroides (Bacteroides) is higher than a threshold value;
(4) The content of bilirubin (bilirubin) is higher than a threshold value;
(5) The content of trimethoprim (trimethoprim) is lower than a threshold value;
(6) The content of stearamide (stearamide) is lower than a threshold value;
(7) The content of phenylalanine (phenylalanine) is higher than a threshold value;
(8) The content of lysophosphatidylethanolamine 17 (PE lyso 17).
The embodiments of the present invention have been described in detail with reference to the drawings, but the present invention is not limited to the embodiments, and various changes can be made within the knowledge of those skilled in the art without departing from the gist of the present invention. Furthermore, the embodiments of the present invention and the features of the embodiments may be combined with each other without conflict.
SEQUENCE LISTING
<110> Shenzhen citizen hospital
<120> IgAN intestinal flora marker, igAN metabolite marker and application thereof
<130> 1
<160> 2
<170> PatentIn version 3.5
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cctacgggng gcwgcag 17
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ggactachvg ggtatctaat 20
Claims (3)
1. Use of an agent for detecting the level of an IgAN metabolite marker comprising trimethoprim, stearamide and cis-9,10-epoxystearic acid in the preparation of an IgAN prognostic reagent.
2. A computer-readable storage medium having stored thereon computer-executable instructions for performing a method comprising:
(1) Obtaining the content of IgAN metabolite markers in a subject sample, wherein the IgAN metabolite markers comprise trimethoprim, stearamide and cis-9,10-epoxystearic acid;
(2) And comparing the content with a preset threshold value, and judging the IgAN prognosis risk according to the comparison result.
3. A system for assessing a subject's risk of prognosis of IgAN, comprising:
detecting means for determining the amount of IgAN metabolite markers in the subject sample, said IgAN metabolite markers comprising trimethoprim, stearamide and cis-9,10-epoxystearic acid;
and the comparison device is used for comparing the content with a preset threshold value and judging the IgAN prognosis risk according to the comparison result.
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